The Gram-negative bacterium Vibrio cholerae is classified as a Category B food- and water-borne pathogen, causing the acute, severe, diarrheal disease known as cholera. Unfortunately, cholera still remains a serious health threat to developing countries with approximately 3-5 million cases occurring annually. V. cholerae is a normal inhabitant of aquatic environments, belonging to the free-living bacterial flora in estuarine areas. Although approximately 200 recognized O serogroups are known, only V. cholerae strains bearing the lipopolysaccharide (LPS) somatic antigens O1 or O139 have been associated with cholera pandemics. As is the case with most Gram-negative bacteria, the LPS of V. cholerae is composed of three distinct regions the membrane associated lipid A domain, a short core oligosaccharide, and the O-antigen polysaccharide. Although the lipid A domain is an essential component of Gram-negative bacterial membranes and is synthesized via a conserved pathway, it is a highly diverse molecule. Pathogenic bacteria modify the lipid A domain of their LPS in response to their surrounding environment. Since lipid A is the bioactive portion of LPS, these modifications can have a profound impact on disease, by altering LPS recognition via the innate immune receptor complex, TLR4/MD-2. Additionally, alteration of the lipid A structure can impact the outer membrane permeability barrier, and bacterial resistance to host antimicrobial peptides. Our overall objective is to understand how alterations in the structure of LPS located on the bacterial surface promote survival of V. cholerae both in the aquatic environment and in the human host. This proposal will focus on defining structural alterations of V. cholerae lipid A in response to the bacterium's extracellular environment and on the enzymatic mechanisms required for this process. Structural alterations of V. cholerae lipid A will be monitored under diverse growth conditions that mimic conditions found either in the aquatic environment or in the small intestine. Completion of the aims below will significantly increase our understanding of the bacterial mechanisms contributing to cholera and possibly provide targets for the development of novel therapies and improved vaccines. The specific aims of the current proposal are: (i) structural analysis of V. cholerae lipid A species; (ii) environmental regulation of V. cholerae lipid A structure; (iii) enzymatic modification of V. cholerae lipid A; and (iv) Toll-like receptor mediated immune activation by V. cholerae LPS.